Isolation and E2 Treatment of Mouse OSE

OSE cells were isolated from mouse ovaries, maintained, and treated with E2 as previously described [4 (link),52 (link)]. OSE were maintained at 37°C in OSE media that consists of α-MEM media (Corning) with 5% fetal bovine serum, 2ng/mL epidermal growth factor (Sigma), and 0.01mg/mL insulin-transferrin-sodium selenite supplement (Roche). For E2 treatment of OSE, cells were seeded and allowed to normalize to hormone-free media consisting of 5% charcoal-stripped fetal bovine serum in phenol red-free DMEM-F12 media (Sigma) for 48 hours before treating with 100nM E2 (Sigma). An equivalent volume of 100% EtOH (vehicle) was added to control dishes for a final concentration of 0.0002% EtOH. Media was refreshed every 3–4 days and collected for single cell RNA-sequencing or immunofluorescent staining 15 days after E2 treatment. All control and E2-treated OSE cell experiments analyzed in this study are after 15 days in culture.